... technique (Labtec. Notebook Curvefit@, Laboratories Technologies Corp.) ... extended from the surface to ca 9 m, a subsurface fluorescence ..... 0.9-13.3 (8.9).
Vol. 69: 103-116, 1991
MARINE ECOLOGY PROGRESS SERIES Mar. Ecol. Prog. Ser.
Published January 10
Effects of irradiance on nitrogen uptake by phytoplankton: comparison of frontal and stratified communities Department of Oceanography, University of British Columbia. Vancouver, British Columbia, Canada V6T 1W5
* Marine Biology Research Division 0202, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093, USA R. M. Parsons Laboratory. Department of Civil Engineering, Massachusetts Institute of Technology. Cambridge, Massachusetts 02139, USA
ABSTRACT: Rates of NOT and urea uptake by phytoplankton in the shallow and deep chlorophyll layers of the Strait of Georgia, British Columbia (Canada) were measured, over a gradient of photosynthetic photon flux densities (PPFD),using the 15N tracer technique. The results of these experiments could be fitted with a hyperbolic function similar to the Michaelis-Menten equation and included a term for dark uptake. Half-saturation constants (KLT) for light-dependent uptake of urea and NOT ranged from 0 to 14 % of the surface PPFD, and dark uptake of both urea and NOT ranged from 0 to 58 % of the uptake at saturating PPFD. Although the importance of dark uptake increased with increased N limitation, the dramatic difference in phytoplankton community composition between the N-replete frontal waters and the N-depleted stratified waters precludes a simple explanation of PPFD effect@)on N uptake based solely on phytoplankton N status. The results of this study are compared to those reported for other aquatic systems.
INTRODUCTION
In most marine and freshwater systems, the uptake of nitrogenous nutrients by phytoplankton is related to the availability of the nutrients (e.g. MacIsaac & Dugdale 1969, Probyn 1985) and to photosynthetic photon flux density (PPFD) (e.g. MacIsaac & Dugdale 1972, Priscu 1984). The dependence of nitrogen uptake upon PPFD has been described by a rectangular hyperbola similar to the Michaelis-Menten formulation in many marine (e.g. MacIsaac & Dugdale 1972, Slawyk et al. 1976 ) and freshwater (e.g. Priscu 1984, Whalen & Alexander 1984) communities. Although nitrogen uptake and assimilation by phytoplankton are dependent upon PPFD as an energy source, either directly or indirectly through photosynthesis, the exact biochemical mechanism(s) by which light regulates nitrogen
Addressee for correspondence: W. P. Cochlan in La Jolla, California O Inter-Research/Printed in Germany
metabolism remains unresolved (e.g. see review by Syrett 1981). The presence of N03-activated ATPase, apparently located within the cell membranes of a number of marine phytoplankters (Falkowski 1975a,b), provides a physiological basis for the coupling between light and NO: uptake, and specific ATPases probably exist for the uptake of NH; and urea as well. The energy (ATP) generated by photophosphorylation is ultimately required for the functioning of these uptake enzymes (permeases) and may also drive the reactions of NH4f (GS/GOGAT) and urea (UAL-ase) assimilation. In addition, the photogeneration of reductants NAD(P)H and reduced ferredoxin will drive the reduction of NO:, NO; and the GOGAT reaction of NH4+ assimilation. Other possible interactions of light with inorganic nitrogen metabolism of phytoplankton are discussed in detail by Syrett (1981). Numerous culture studies have demonstrated that Ndeprived phytoplankton have greater dark uptake rates of N than do N-replete phytoplankton (e.g. Syrett 1962, Eppley & Coatsworth 1968, Thacker & Syrett
104
Mar. Ecol. Prog. Ser. 69: 103-116, 1991
1972, Rees & Syrett 1979), suggesting a lesser light dependence on N uptake during N stress. This, together with field studies which show that deep-living phytoplankton sustain substantial N uptake velocities with little or no light (e.g. Conway & Whitledge 1979, Nelson & Conway 1979, Priscu 1984), suggests that both light exposure and nutritional history of phytoplankton may be important in determining their ability to sequester nitrogen, and that these controlling factors may differ for the various forms of nitrogen. Shallow sea fronts, located at the boundary between stratified and vertically mixed regimes (see reviews by Denman & Powell 1984, LeFevre 1986), are generally areas of high primary productivity (e.g. Pingree et al. 1975, Parsons et al. 1981, 1983, Holligan et al. 1984). These regions are characterized by having surface water with high phytoplankton biomass and measurable concentrations of nitrate, and a shallow pycnocline which extends to the surface at the frontal boundary (e.g. Simpson & Pingree 1978). A surface transect normal to a frontal boundary progresses from high concentrations of dissolved NOT on the well-mixed side to N-depleted, stratified waters, and thus represents a gradient of both nitrogen and light availability and consequently of phytoplankton physiological states. Moreover, the nitrogenous nutrition of the phytoplankton would likely differ along such a transect. In the N-impoverished waters, the N demands of phytoplankton are supplied by reduced N forms such as NH: and urea from regenerative processes, whereas in N-rich areas, nitrogen compounds are generally utilized at rates proportional to their availability (e.g. Dugdale & Goering 1967, McCarthy et al. 1977). The experiments presented in this study were conducted in the Strait of Georgia, a partially enclosed coastal basin on the west coast of Canada (see reviews by Harrison et al. 1983, LeBlond 1983),where several tidally induced frontal regions have been previously described (Parsons et al. 1981, Price et al. 1985). The influence of PPFD on the uptake of NO: and urea by phytoplankton from nitrate-replete frontal water and nitrate-depleted stratified water was examined, and the dependence of N uptake on PPFD by the phytoplankton from the subsurface chlorophyll maximum of these 2 distinct areas was compared. Simulated in situ experimental conditions were attempted in order to obtain a better understanding of the true NO: uptake response to PPFD in these physically and chemically distinct environments. Most previous studies of the effect(s) of PPFD on N uptake by phytoplankton have employed saturating enrichments of isotopically labelled N forms (e.g. MacIsaac & Dugdale 1972, Priscu 1984, Mitamura 1986), and reported uptake rates may reflect the effects of both PPFD and N concentration.
MATERIALS AND METHODS
General. Nitrogen uptake experiments were conducted in the Strait of Georgia, B.C., Canada, aboard the CSS 'Vector' during July and August 1984; station locations and names are shown in Fig. 1. Between 14:OO and 15:OO h PDT (Pacific Daylight Time), water samples were collected using 5 1 PVC Niskin bottles, from just below the sea surface (0 to 1 m) and from depths corresponding to the deep chlorophyll maximum (DCM).The shallow sampling depth represents a light environment of ca 80 to 100 % surface PPFD (I,) for each station, whereas the DCM depth corresponds to ca 8, 3 and 2 O/O I, for Stations T14, A5 and T8, respectively. Samples were shielded from direct sunlight during transfer to 10 1 Nalgenem carboys and taken into the ship's laboratory. Subsamples for nutrient analyses were removed with an acid-washed syringe and gently filtered through combusted (460 "C for 4 h) Whatman GF/F filters (mounted in 25 mm Millipore Swinexm filter holders) into acid-washed polyethylene bottles. Nitrate plus nitrite (NO: + NOT) and ammonium (NH:) were measured immediately with a Technicon AutoAnalyzerm 11, following the procedures outlined in Wood et al. (1967) and Slawyk & MacIsaac (1972),respectively. Urea was determined by the diacetyl monoxime thiosemicarbizide technique described by Price & Harrison (1987). Samples for chlorophyll a (chl a) [coefficient of variation (CV) = 4.4 4.1 %; 5 sample pairs] were collected on Whatman GF/F filters and stored frozen in a desiccator. Chl a was extracted in 90% acetone overnight and analyzed by in vitro fluorometry (Strickland & Parsons 1972) using a Turner Designs Model 10 fluorometer. Particulate organic carbon (POC) and nitrogen (PON) (CV = 5.2 4.8 % and 3.8 f 4.1 % respectively; 7 sample pairs), collected on combusted Whatman GF/F filters, were stored similarly and analyzed later after drylng (24 h at < 60 "C) with a Perkin Elmer Model 240 elemental analyzer, using the dry combustion method described by Sharp (1974). At each station continuous vertical profiles (0 to 20 m) of temperature, salinity, fluorescence and NO: NO; were run prior to the bottle casts. Temperature and salinity were determined with an Interocean 514A NO, CSTD system; in vivo fluorescence and NO: concentrations were obtained from pumped samples (mRoy FR162-144 diaphragm pump, flow rate ca 1 1 min-l) and measured with a Turner Model 111 fluorometer (equipped with a flow-through cell) and a Technicon AutoAnalyzerm 11, respectively. These data were logged onto a computer and plotted in real-time using a custom software programme which compensates for time lags in pumping and machine analyses (Jones et al. in press). Incident solar irradiance (PAR,
+
+
+
+
Cochlan et al.: Irradiance effects on phytoplankton N uptake
105
Fig. 1. Station locations for nitrogen uptake experiments. Frontal (T14), shallow stratified (A5) and deeply stratified (T8) stations in the Strait of Georqia, British Columbia 400 to 700 nm) was monitored continuously with a Lambda Instruments LI-185 light meter that was equipped with a LI-19OSB Surface Quantum Sensor and connected to a chart recorder. Subsurface irradiances were measured with a LI-185B light meter equipped with a LI-192s Underwater Quantum Sensor (2n). Phytoplankton samples (250 ml) were preserved in acid Lugol's solution (Parsons et al. 1984) and stored in the dark until counting. Subsamples (10 ml) were settled (24 h) and counted on a Wild inverted microscope following Utermohl (1958). Experimental. Within 1 h of collection, water samples from each depth were transferred under reducedlight conditions to 500 m1 Wheaton glass bottles with teflon-lined caps. Nitrate and urea uptake rates were measured using the stable isotope 15N (Kor Isotopes) as a tracer (Dugdale & Goering 1967).For the urea experiments, C 0 ( 1 5 N ~ 2(99 ) 2 atom %) was added to bring the final 1 5 concentration ~ to either 2 or 4 yg-at. N1-l. In the nitrate experiments, Na15N03 (99 atom %) was added in concentrations of either 0.05 pg-at. N 1-l or < l 0 % of the ambient NO; NO: concentration. These enrichments were not always true tracer additions (usually defined as 5 10 % of ambient), but the term 'tracer' will be used here to distinguish the low 15N03 enrichments from the saturating enrichments
+
associated with the urea uptake experiments. Following enrichment, bottles were immediately mixed and placed within neutral-density screening to simulate the following PPFDs: 95, 55, 31, 10, 3.4, 1.1 and 0 % I,. The screen material used in the incubators was calibrated with a Biospherical Instruments QSL-100 4n sensor placed within an incubation bottle. The 0 O/O PPFD was achieved by multiple wrappings of the bottle with black tape. Incubations were conducted at in situ temperatures (+ 1.5 "C) under natural light in clear, Plexiglasmdeck incubators. Clear, cloudless skies prevailed throughout the cruise, resulting in virtually identical ambient light conditions during each experiment. Samples from the surface waters were cooled with flowing surface seawater, while deeper samples were incubated in a separate, temperature-controlled incubator. Incubations were terminated after 2 to 4 h by filtration (pressure differential < 125 mm Hg) onto combusted Whatman GF/F filters, placed into plastic petri dishes, and stored frozen in a desiccator. Based on the ambient dissolved nitrogen concentration, the initial particulate nitrogen concentration, and the final 15N atom percentage in the particulate fraction, it was calculated that a mean (-+ SD) of 24.1 f 15.3 % and 8.5 k 5.2 % of the NO3 and urea, respectively, in solution was incorporated into particulate material during
Mar. Ecol. Prog. Ser. 69: 103-116, 1991
106
Kinetic parameters of uptake. The kinetic constants for NO: and urea uptake with respect to irradiance were obtained by a direct fit of the data to a modified Michaelis-Menten hyperbola using a computerized, iterative, non-linear least-squares technique (Labtec Notebook Curvefit@,Laboratories Technologies Corp.). The Michaelis-Menten equation, modified to account for dark uptake, describes uptake over the hyperbolic part of the curve (MacIsaac & Dugdale 1972) and is as follows:
the incubation period. Substrate exhaustion was therefore not considered a problem in the experiments of this study. Nitrogen in the particulate samples was converted to dinitrogen gas (NZ)by the micro-Dumas dry combustion technique (La Roche 1983) and then analyzed for I5N enrichment with a JASCO Model N-150 emission spectrometer (Fiedler & Proksch 1975). Generally, each sample was scanned 6 times (minimum of 3 times), and the average 15N/14Npeak height ratio was used in the calculation of the atom percent I5N (specific activity) in the particulate material. Automatic selection of peak heights during scans, and isotopic ratio calculations, were performed utilizing in-house software (Jones unpubl.) on an IBM-compatible PC interfaced with the spectrometer. The emission spectrometer was routinely calibrated with a series of pure N2 gas standards supplied by JASCO of known I5N enrichment. Nitrogen uptake rates were calculated using Equation 7 of Dugdale & Wilkerson (1986) (equivalent to Equation 5 of Collos 1987), which corrects for changes in PON during the incubation period. Corrections were not made for isotopic dilution from remineralization of 14N-urea during the incubation (Hansell & Goering 1989), as this correction would probably be negligible given the large amount of 15N-labelled urea added to the bottles. Specific rates of nitrogen transport were calculated by dividing the volumetric rates by the phaeophytin-corrected chl a concentration at the beginning of the experiments. Although chl a per cell may vary with depth due to PPFD differences, it was chosen as the normalization parameter because it absorbs the light necessary to fuel cellular transport mechanisms. Use of chl a specific uptake rates also facilitates comparison with previously published studies on chl a normalized nitrogen and carbon uptake vs irradiance. TEMPERATURE (OC)
0
.2
0
5
.4 10
.6
.8
1.0 0
.l
.2
15
20
25 0
5
10
RELATIVE FLUORESCENCE
,
I
v = VD + V',,,
J
-
[Klr
+
where V = total uptake of N per unit of chlorophyll; VD = dark value of V; I = integrated average PPFD during the incubation period; V',,, = maximum N uptake per unit chlorophyll at saturating PPFD; and KLT (the halfThe saturation constant for light) = PPFD at 0.5 V',,,. assumption is made that dark uptake is a constant at all light levels. Only data obtained from non-photoinhibitory PPFDs were used in this analysis.
RESULTS AND DISCUSSION
General description of stations The vertical profiles of temperature, salinity, relative NO; concenin vivo chl a fluorescence and NO: tration, for the 3 stations at which N uptake vs PPFD experiments were conducted, are presented in Fig. 2. The diagnostic features of the frontal water (Station T14) included a weak thermocline and halocline which extended from the surface to ca 9 m, a subsurface fluorescence maximum layer (ca 5 to 8 m), a nitracline which extended to the surface, and relatively high NO: + NOT concentrations throughout the water column.
+
,
SALINITY
(%.l
28
29
24
25
16
18
10
12
.3
.4
.5
0
15
20
25
0
NITRATE+ NITRITE
26
27
28
29 0/,
14
16
18
20°C
.l
.2
.3
.4
.5 Rel. Fluor.
5
10
15
20
25pgatN1-'
.
(ppat N I - ' )
Fig. 2. Depth profiles of temperature (T), salinity (S), in vivo fluorescence (F) and nitrate plus nitrite concentration (N) for the 3 stations sampled (T14: frontal; A5: shallow stratified; T8: deeply stratified). The horizontal dashed line denotes the depth of 1% surface PPFD (photosynthetic photon flux density)
Cochlan et al.: Irradiance effects on phytoplankton N uptake
107
Table 1. Initial environmental conditions of seawater collected for N-uptake vs irradiance experiments. PDT: Pacific Daylight Time; Chl a: chlorophyll a; PON: particulate organic nitrogen; POC: particulate organic carbon; (-): not determined Station and location
Description
Date (1984)
Starting Sample time of depth incubation (m) (PDT)
Nitrogen conc. NOa-
Chla (kg I-')
Urea NH4+a (pg-at.N 1-l)
PON (~g-at. N1-l)
POC (kg-at. Cl-l)
T14
4g053'24"N 125"05'06W
Frontal
27 Jul
15:30h
0 8
6.02 15.05
-
0.23 0.21
1.29 2.28
5.28 6.96
43.1 40.6
A5
49"53'02"N 125"05'48W
Shallow stratified
30 Jul
14:30 h
0 15
0.2 pm) could assimilate up to half the quantity of methylamine as could phytoplankton (>3 pm) and that decreasing PPFD increased the methylamine uptake by surface communities of bacteria relative to phytoplankton. By comparing the nitrogen uptake rate of 1 5 ~ ~ 4with + the rate of 14c02 incorporation into protein, Laws et al. (1985) concluded that heterotrophic N
12
NE Pacific Ocean
Strait of Georgia, B.C. Frontal Surface stratified Bottom stratified
Western Irish Sea Surface stratified Mixed & bottom strat.
Auke Bay, Alaska
ca 2.5 ca 4.5
< 1.0
> 1.0
6-15 < 0.05 7-20
Strait of Georgia, B.C. Frontal Stratified
Washington coast (USA)
3.0-4.6 20
> 20
>2 10 > 10
>2 < 0.1
N Pacific Ocean Northern (51-57) Tropical/subtropical ( J 9 J 2 3 )
Upwelling NW Africa
> 10 0-1.0
Ambient N o ? conc. (pg-at. N 1-l)
N Pacific Central Gyre 50" N, 155" W 40" N, 155" W
Oceanic N Atlantic Gyre (Sargasso Sea)
Area
(0.21)
-
0.0-0.057 (0.005) 0.14-0.47 (0.27)
-
0.25-0.28 (0.27) 0.48-0.51 (0.50) 0.00-0.49 (0.25)
0.00-0.08 (0.03) 0.00-0.18 (0.09)
0.06-0.57 (0.17) 0.18-1.7 (0.47) 0.2-1.0 (0.7) 0.0-0.2 (0.10) 0.60-0.86 (0.73)
0.88-1.2 (1.0) 0.27-1.0
0.01-0.67 (0.16) 0.02
0.00-0.02 (0.07)
0.0-0.02 (0.09)g
0.0-0.097 (0.024) 0.075-0.32 (0.22)
0.0-0.63 (0.30) 0.92
(0.30)
NO3
(0.59)
(0.43) 0.086-0.096 (0.091) 0.41-0.55 (0.48)
-
0.37-0.39 (0.38) 0.52-0.58 (0.55)
0.47-1.1 (0.67) 0.37-1.3 (0.72)
Turley (1985)~
Kanda et al. (1989)'
Dortch & Poste1 (1989)
-
(0.38)
l 0.58-0.77 (0.68) 0.15-0.16 (0.16)
Present study
Price et al. (1985)
Conway & Whitledge (1979) Dugdale & Goering ( 1 9 6 7 ) ~ * ~ Dortch & Maske (1982)
0.4-1.3 (0.7) 0.0-0.4 (0.26) -
Glibert et al. (1982)C*g Ronner et al. (1983)' Kristiansen & Lund (1989)'
Nelson & Conway (1979) MacIsaac (1978)~
Nelson & Conway (1979)
Cochlan (1989)
Kanda et al. (1985)
Hattori & Wada (1972)
Dugdale & Goering (1967)a
Source
Paasche & Erga (1988)a
0.00-0.81 (0.36) 0.06-0.66 (0.36)
0.3-0.5
-
-
0.0-0.19 (0.093) 0.12-0.50 (0.26)
-
-
Urea
-
0.30-0.47 (0.38) 13.0-75.0
0.10-1.46 (0.49) 0.34
0.05-0.57 (0.36)
-
0.057-0.27 (0.17) 0.17-0.53 (0.34)
0.38-2.0 (0.83) 0.78-1.5 (1.2)
-
P,/P, or V D N L NH4
Table 6. Summary of literature values of dark:light (dark incubation bott1es:clear incubation bottles) specific (VD/VL)or absolute (pD/pL)nitrogen uptake rates, determined during daytime, in natural phytoplankton communities (values given are ranges, with means in parentheses)
NO3
0.26
-
-
50.07
L. Nakanuma (Japan)
L. Biwa (Japan)
L. Kasumigaura (Japan)
-
0.0-0.48 (0.21)
11.1
G c a 2.5
-
G15
Amazon River (Brazil)
Castle L. (California, USA)
Flathead L. (Montana, USA)
L. Ontario (Canada)
(0.50)
(0.55)
'
0.30-0.60 (0.40)
0.44-0.82 (0.64)
0.54
0.11-1.0 (0.49)
0.47 0.83
-
Liao & Lean (1978)g
Dodds & Priscu (1989)'
Priscu (1984)'
-
Fisher et al. (1988)
Prochazkova et al. (1970)
Priscu (1989)~
Whalen & Alexander (1984)
Toetz & Cole (1980)a2e
Takamura et al. (1987)
Mitamura & Saijo (1986)
Miyazaki et al. (1985)~
Berman et al. (1984)
Fisher et al. (1988)
+
Fisher et al. (1982)
McCarthy et al. (1982)a
I
,
Source
-
0.71
0.51
-
0.13-0.67 (0.34) 0.33-0.43 (0.38)
-
-
-
Urea
a
'
Values calcul&d&i~ l/(VLND)or l/(pL/pD)from reported values of VL/VDor pL/pD Values are p,/~:, where N$, is the chlorophyll-specific transport rate at optimal PPFD Values estimated from figiires g Experiments utilized 24 h incubations over natural light/dark cycle C Values reported in text, no data available h Average values reported Values calculated from turnover times Values calculated as VD/(VD V,') Light rates determined at ambient N conc., dark rates determined a t s&iirating N conc. *tTlcnoAagell$e bloom * * Microcystis bloom
0.02-0.30 (0.14)
0.20
0.00-0.32 (0.16)
0.05-0.38 (0.11)
> 35
ca
